Lázcoz Ripoll, Paula

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https://academica-e.unavarra.es/handle/2454/49387

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Lázcoz Ripoll

First Name

Paula

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Ciencias de la Salud

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1172611

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7324

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Now showing 1 - 6 of 6

Open Access
Changes in gene expression profiling of apoptotic genes in neuroblastoma cell lines upon retinoic acid treatment
(Public Library of Science, 2013) Celay Leoz, Ion; Blanco Luquin, Idoia; Lázcoz Ripoll, Paula; Rotinen Díaz, Mirja Sofia; Castresana, Javier S.; Encío Martínez, Ignacio; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
To determine the effect of retinoic acid (RA) in neuroblastoma we treated RA sensitive neuroblastoma cell lines with 9-cis RA or ATRA for 9 days, or for 5 days followed by absence of RA for another 4 days. Both isomers induced apoptosis and reduced cell density as a result of cell differentiation and/or apoptosis. Flow cytometry revealed that 9-cis RA induced apoptosis more effectively than ATRA. The expression profile of apoptosis and survival pathways was cell line specific and depended on the isomer used.
Open Access
Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines
(Spandidos Publications, 2010) Schiapparelli, Paula; Enguita Germán, Mónica; Balbuena, Jana; Rey, Juan A.; Lázcoz Ripoll, Paula; Castresana, Javier S.; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, 9/07
Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of all childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines. RT-PCR and flow cytometry were performed in order to analyze different kinds of ‘stemness genes’ such as: NESTIN (NES), CD133, SOX-2, BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). In addition, glial and neuronal markers such as NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Epigenetic changes within the CD133 (Prominin-1) gene promoter were also analyzed. Neuroblastoma cell lines showed a particular pattern of expression, suggesting the presence of an immature cancer stem cell-like subpopulation. The CD133 protein, commonly used to enrich putative cancer propagating stem cell-like populations in different kinds of solid tumors, presented a half-methylated DNA state in 7 of the 12 neuroblastoma cell lines analyzed. An increase in RNA and protein levels of CD133 was achieved following demethylation by assays using 5-aza-2'-deoxycytidine (5-Aza-dC). Since cancer stem cells are believed to be responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Moreover, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.
Open Access
Promoter methylation of RASSF1A associates to adult secondary glioblastomas and pediatric glioblastomas
(Hindawi / Wiley, 2012) Muñoz, Jorge; Inda, María del Mar; Lázcoz Ripoll, Paula; Zazpe Cenoz, Idoya; Fan, Xing; Alfaro, Jorge; Tuñón Álvarez, Teresa; Rey, Juan A.; Castresana, Javier S.; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent.
Open Access
Frequent promoter hypermethylation of RASSF1A and CASP8 in neuroblastoma
(BioMed Central, 2006) Lázcoz Ripoll, Paula; Muñoz, Jorge; Nistal, Manuel; Pestaña, Ángel; Encío Martínez, Ignacio; Sáez Castresana, Javier; Ciencias de la Salud; Osasun Zientziak
Background: Epigenetic alterations and loss of heterozygosity are mechanisms of tumor suppressor gene inactivation. A new carcinogenic pathway, targeting the RAS effectors has recently been documented. RASSF1A, on 3p21.3, and NORE1A, on 1q32.1, are among the most important, representative RAS effectors. Methods: We screened the 3p21 locus for the loss of heterozygosity and the hypermethylation status of RASSF1A, NORE1A and BLU ( the latter located at 3p21.3) in 41 neuroblastic tumors. The statistical relationship of these data was correlated with CASP8 hypermethylation. The expression levels of these genes, in cell lines, were analyzed by RT-PCR. Results: Loss of heterozygosity and microsatellite instability at 3p21 were detected in 14% of the analyzed tumors. Methylation was different for tumors and cell lines (tumors: 83% in RASSF1A, 3% in NORE1A, 8% in BLU and 60% in CASP8; cell lines: 100% in RASSF1A, 50% in NORE1A, 66% in BLU and 92% in CASP8). In cell lines, a correlation with lack of expression was evident for RASSF1A, but less clear for NORE1A, BLU and CASP8. We could only demonstrate a statistically significant association between hypermethylation of RASSF1A and hypermethylation of CASP8, while no association with MYCN amplification, 1p deletion, and/or aggressive histological pattern of the tumor was demonstrated. Conclusion: 1) LOH at 3p21 appears in a small percentage of neuroblastomas, indicating that a candidate tumor suppressor gene of neuroblastic tumors is not located in this region. 2) Promoter hypermethylation of RASSF1A and CASP8 occurs at a high frequency in neuroblastomas.
Open Access
In vitro assessment of the role of p53 on chemotherapy treatments in neuroblastoma cell lines
(MDPI, 2021) Blanco Luquin, Idoia; Lázcoz Ripoll, Paula; Celay Leoz, Ion; Castresana, Javier S.; Encío Martínez, Ignacio; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2 /p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etopo-side, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.
Open Access
Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR
(BioMed Central, 2008) Lorente, Aiala; Mueller, Wolf; Urdangarín, Edurne; Lázcoz Ripoll, Paula; Deimling, Andreas von; Sáez Castresana, Javier; Ciencias de la Salud; Osasun Zientziak
Background: We present two melting curve analysis (MCA)-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, i. e. bisulfite sequencing and standard MSP. Methods: The promoters of the RASSF1A (3p21.3), BLU (3p21.3) and MGMT (10q26) genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing. Results: Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent. Conclusion: We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.